The principal goal of this project is to characterize the molecular mechanisms by which the RNA genome of HIV-1 is exported from the nucleus of an infected host cell. HIV-1 RNA nuclear export requires the formation of a defined transport complex, composed of the viral Rev protein, the Rev-response element (RRE), and the host proteins, CRM1 (also known as XPO1 or Exportin 1) and Ran. The RRE is a 250-nucleotide segment of the genome, which is predicted to have a well-defined tertiary structural fold. Despite almost three decades of intense effort, the structures of the RRE and of the transport complex are still unknown. This lack of structural data significantly hinders a molecular level understanding of this critical step in HIV-1 replication. We therefore propose to: (1) Determine the structure of the RRE. Novel experimental approaches will be developed and applied in order to obtain homogeneous preparations of the RRE in multimilligram quantities. Novel RNA crystallization aids will be developed to facilitate RRE crystallization and structural analysis byx-ray crystallography. Optimization of constructs will be performed in an assay-guided manner. (2) Reconstitute the Rev/RRE complex using purified RNA and protein subunits. Various reconstitution methods will be applied, with the goal of obtaining samples of homogeneous stoichiometry and composition. Biochemical and structural analysis will be performed in conjunction with virological and cell biological assays. Overall, the proposed experiments will directly test alternative models of RRE structure, define the mechanisms that control the subunit stoichiometry of the RNA transport complex, and begin to probe molecular details of how the Rev/RRE system co-opts the host cell's nuclear export machinery. RNA transport is an essential, rate-limiting step in HIV-1 replication, and these studies are likely to suggest new strategies to inhibit the virus.